In the field of molecular biology it is often desirable to transfect cells to express multiple genes. Classical methods for achieving this have relied upon integration of multiple genes into one or more chromosomal loci. The sites of gene integration, however, are random, and the number and ratio of genes integrating at any particular site are unpredictable. Therefore every transfected cell is unique. Furthermore, expression of the integrated genes may be subject to unpredictable position effects, e.g., those caused by adjacent chromosomal sequences. In some cases, amplification of the genes of interest is required in order to achieve adequate expression levels. As a result, it is normally necessary to screen many clonal cell populations to obtain a cell line in which all of the desired genes are expressed at an appropriate level. This procedure of transfection, selection and analysis of numerous clonally derived cell lines expressing the multiple genes can take many months.
For example, simultaneous transfection of HEK293 cells with vectors encoding the .alpha.1, .alpha.2, and .beta.3 subunits of human calcium channel has been carried out to obtain fully functional expression of that multi-subunit protein from chromosomally incorporated copies of the transfected genes. However, obtaining cells that functionally express all three subunits requires extensive screening of cell populations, while finally obtaining very few colonies (Buchert et al., Biotechniques (UNITED STATES) 23, 402-407, 1997).
Non-integrating, autonomously replicating episomal vectors have been used to transform cells to express a gene of interest. In particular, the Epstein Barr Virus (EBV) Nuclear Antigen 1 (EBNA 1) has been used to stably maintain a plasmid containing an EBV origin of replication (oriP) in primate cells (Reisman, D. et al, Mol. Cell. Biol. 5: 1822-1832, 1985; Yates, J. L. et al., Nature 313:812-815, 1985). The plasmid is maintained in an episomal state, i.e., it is not integrated into the chromosome.
Transfection of cell lines that express EBNA 1 can be advantageous since the ability of such cells to stably maintain an episomal construct can be enhanced by several orders of magnitude, and stable cell lines can be generated in as little as two to three weeks. For example, HEK cells that stably express EBNA 1 have been transformed with plasmids containing the EBV origin of replication, and the gene encoding CRHR1 (corticotropin releasing hormone receptor subtype I). The resulting cell lines have been found to stably express high levels of CRHR1. (Horlick et al., Prot. Exp. And. Purific. 9:301-308, 1997.)
Similarly, U.S. Pat. No. 4,686,186 describes transfecting cells with a single plasmid containing the EBV oriP, the EBNA 1 gene, and a gene encoding a protein of interest (U.S. Pat. No. 4,686,186).
Expression of multiple genes on a single plasmid, however, can result in promoter occlusion. (Greger, I. H. et al., Nuc. Acid Res. 26(5): 1214-1301, 1998; Kadesch, T. et al., Mol. Cell. Biol. 6(7): 2593-2601, 1986). In cases of promoter occlusion, one strong promoter can bind most or all of the transcription factors in its immediate vicinity, thereby limiting transcription from other promoters present in cis on the same plasmid. This, in turn, causes the expression of multiple genes of interest on a single episome to be unpredictable and often problematic (Horlick et al., 1997). The EBNA1/oriP expression system has not, therefore been widely used to express multiple genes of interest.
Currently, each cell type for which episomal expression is desired is typically first transfected with an integrating copy of the gene encoding EBNA 1. Since developing cell lines that constitutively express EBNA 1 from an integrated gene is time consuming, current methods are somewhat limited in their applicability to different cell lines. Programs for mass screening of compound libraries require use of many types of cell lines, and producing EBNA 1 producing strains of each type by this method requires an extensive effort.
Alternately, episomes that already carry the EBNA 1 gene and a gene of interest in cis on the same episome can be used to transfect cells. Commercial vectors such as pCEP4 (Invitrogen) are available for this purpose. However, current vectors in which EBNA 1 is carried by the episomal construct in cis do not contain a known promoter for driving expression of EBNA 1. Rather, it is believed that transcription of the EBNA 1 gene occurs from a fortuitous promoter situated in or near an amp resistance marker that is located a few hundred nucleotides upstream from the EBNA 1 start codon. This fortuitous promoter, however, is not sufficiently recognized by differing cell types to consistently express EBNA-1 with sufficient speed and abundance to sustain the replication and maintenance of the episome (before it is otherwise lost from the cell). Therefore, currently available episomal vectors containing the EBNA 1 gene in cis do not appear to provide sufficient reliability for use in a wide variety of cell types. Furthermore, adding a strong promoter to these episomes to express the EBNA-1 gene in cis would, under certain circumstances, result in promoter occlusion.
Multiple plasmids have been used to transform bacterial cells. However, to the inventors's knowledge, transfection of eukaryotic cells with multiple plasmids has not been described. Furthermore, it has not been known whether transfecting a eukaryotic cell with a second or third episome would disrupt an already resident first episome. For example, it has not been known whether transfection of separate episomal constructs into eukaryotic cells would result in stable maintenance of both constructs, or in efficient transcription or translation of separate genes contained in both constructs.
There is therefore a need for a method that allows rapid production of eukaryotic cells that stably express multiple genes.
There is also a need for a method that allows rapid production of stable cell lines of varying types that express a gene of interest.